Alterations in intra-pituitary TRH content have been produced in animals treated with PTU and T4. In animals treated with PTU, TRH and TSH declined pari passu. In animals given T4 acutely following PTU treatment, TSH and TRH rose concomitantly. TRH has been identified in fetal hypothalami grown in organotypic cultures. TRH synthesis was increased with amingphylline. Leucine5-enkephalin activity has now been identified in human hypothalamic and extrahypothalamic CNS sites using a new RIA. It is proposed to extend ongoing studies of TRH metabolism in the intact rat in isolated rat hypothalami CNS sites using a new RIA. It is proposed to extend ongoing studies of TRH metabolism in the intact rat in isolated rat hypothalami, rat adenohypophyses, and in isolated rat pituitary secretory granules. Studies of TRH biosynthesis and secretion will utilize high specific activity 3H-proline as a TRH precursor in conjunction anti-TRH IgG affinity chromatgraphy and high performance liquid chromatography for 3H-TRH purification. Tissue cultures of fetal rat hypothalami will be accomplished with Falcon organ culture grids and Eagles' Minimal Essential Medium. The following new goals are established: 1) isolation of rat pituitary secretory granules by sucrose density gradient ultracentrifugation for immunochemical and biochemical analyses of TRH0and TSH; 2) TRH biogenesis will be examined in organ cultures of rat hypothalami; 3) TRH metabolism will be examined in LAF1 mouse pituitary TSH-secreting tumors; 4) Identification of TRH in human amniotic fluid will be explored in eluates from TRH IgG affinity chromatography.